Not Applicable.
Not Applicable.
(1) Summary of the Invention
The present invention relates to antigen vaccines and methods of use thereof for the treatment of Pythium insidiosum infections, as well as prophylaxis against these infections, in humans and mammals. Further, the present invention relates to a method for preparing the preferred vaccine for the treatment which contains the intracellular and extracellular antigens of Pythium insidiosum. The present invention further relates to a mammal model for evaluating Pythium insidiosum vaccines and a method for monitoring the Th1 and Th2 response of a mammal in response to Pythium insidiosum vaccines.
(2) Description of Related Art
Infections caused by fungal and parafungal organisms are occurring with increasing frequency in patients with debilitating illnesses such as leukemia and AIDS, as well as those undergoing immunosuppressive therapy. Within this group of organisms are the traditional pathogenic fungi and a long list of newly recognized emerging opportunistic fungal and parafungal organisms. Among these emerging pathogens is the oomycete Pythium insidiosum, a fungal-like organism in the Kingdom Kromista, Phylum Pseudofungi. Pythium insidiosum is not only physiologically distinct from members of the Kingdom Fungi, but also differs physiologically. This may explain why anti-fungal drugs do not have any effect on pythiosis.
Pythiosis insidiosi particularly occurs in humans and lower animals in the tropical, subtropical, and temperate areas of the world (de Cock, W.A.W., et al., J. Clin. Microbiol. 25: 344-349 (1987)). The disease was first described in the beginning of the 20th century in equids of tropical and subtropical countries such as India and Indonesia as well as the USA. Soon, however, it was evident that the disease not only affected equids but other mammalian species such as humans. In lower animals, infections of the cutaneous tissues, lymphatic vessels, intestines, lungs, and bones have been found. In humans, a deadly arteritis infection, subcutaneous invasion, and keratitis occurs.
The drugs currently available to treat fungal infections have had little or no effect on Pythium insidiosum. Reports of treatment with either amphotericin B or surgery, which are commonly used to treat this disease in humans and lower animals, have indicated that 60% of the patients died of their infections. In cases of arterial invasion in humans, amphotericin B did not eliminate the infection (Rinaldi, M. G., et al., Mycol. Obser. 9:7 (1989); and Thianprasit, M., Trop. Dermathol. 4: 1-4 (1990)), whereas in surgery the main problem has been to determine how much of the infected tissues has to be removed. Thus, relapses are common in surgically treated patients, who must also endure the pain and distress that such an invasive traumatic procedure inflicts on them.
The curative properties of Pythium insidiosum antigens was first noticed when some Costa Rican horses with pythiosis, which had been injected with Pythium insidiosum antigens in a skin test, were cured (Mendoza, L., et al., Equine pythiosis in Costa Rica: report of 39 cases. Mycopathologia 94: 123-126 (1986)). Simultaneously, another vaccine with similar curative properties was successfully used in horses with the disease in Australia (Miller, R. I., Aust. Vet. J. 57:3 77-382 (1981)). These two vaccines have been referred to in the literature as the Mendoza vaccine and the Miller vaccine, respectively (Newton, J. C., et al., The Compendium 15: 491-493 (1993)) Early reports indicated that the antigens used in the Pythium insidiosum-vaccine possessed unique characteristics, somewhat similar to the features of those reported in Trichophyton verrucosum (Gudding R., et al., Can. Vet. J. 36: 302-306 (1994)) and other immunotherapeutic vaccines (Foster, J. S., et al., Vet. Med. Small Ani. Clin. 71: 920 (1976); Pier, A. C., et al., Equine Pract. 15: 23-27 (1993)).
The Miller vaccine is prepared from sonicated hyphal antigens (Miller, R. E., Aust. Vet. J. 57: 377-382 (1981)), while the Mendoza vaccine is prepared from culture filtrate antigens (Mendoza, L., et al., Mycopathologia 94: 123-129 (1986)). Both the Miller vaccine and the Mendoza vaccine have been used to cure early cases of equine pythiosis, i.e., horses with pythiosis-caused lesions of 0.5 months or less in duration; however, neither vaccine can cure horses that are chronically infected with Pythium insidiosum, i.e., horses with pythiosis-caused lesions two or more months old (Mendoza, L., et al., Mycopathologia 119: 89-95 (1992)). While both vaccines have cure rates of about 53% for early cases of pythiosis, the Mendoza vaccine has a longer shelf-life and milder side effects than the Miller vaccine (Miller, R. I., et al., J. Am. Vet. Med. Assoc. 182: 1227-1229 (1983)). The Mendoza vaccine, in addition to its immunotherapeutic properties, also showed some degree of protection against disease caused by Pythium insidiosum. However, this protection was later found to be of short duration (Mendoza, L., et al., Mycopathologia 119: 89-95 (1992)).
In over 15 years of use, the Mendoza vaccine has been shown to be safe and consistently efficacious, curing more than 300 horses with pythiosis-caused lesions of short duration. However, the Mendoza vaccine can only cure early equine pythiosis, not chronic cases of this disease (Mendoza, L., et al., Mycopathologia 119: 89-95 (1992)). Aside from the fact that the Mendoza vaccine can only cure early equine pythiosis cases, nothing was known about the immunogens involved in its curative properties nor the immune mechanisms that triggered the killing of Pythium insidiosum""s hyphae infected tissues.
In a recent study using SDS-PAGE and Western blot analysis, the presence of three immunodominant hyphal proteins were identified (Mendoza, L., et al., J. Clin. Microbiol. 30: 2980-2983 (1992)). While the Western blots revealed that the IgG in sera from horses with active pythiosis recognized most of the proteins of Pythium insidiosum, the Western blots also showed that three protein bands of the 32 kDa, 30 kDa, and 28 kDa were particularly prominent. More significant was the finding that antibodies against these three proteins persisted for long periods of time in the sera from successfully cured horses.
Even though there are two vaccines which can be used to treat pythiosis, there remains a need for a vaccine which can cure pythiosis and prevent infection by Pythium insidiosum. In particular, there is a need for a vaccine that can be used to treat and cure patients who are in a chronic stage of the disease.
The present invention provides antigen vaccines and methods of use thereof for the treatment of Pythium insidiosum infections, as well as prophylaxis against these infections, in humans and mammals. Further, the present invention provides a method for preparing the preferred vaccine for the treatment which contains the intracellular and extracellular antigens of Pythium insidiosum. The present invention further provides a mammal model for evaluating Pythium insidiosum vaccines and a method for monitoring the Th1 and Th2 response of a mammal in response to Pythium insidiosum vaccines.
Therefore, the present invention provides a method for treatment of pythiosis in a human patient having pythiosis or prophylaxis against pythiosis which comprises (a) providing a vaccine consisting of intracellular cytoplasmic antigens separated from disrupted cells of Pythium insidiosum and extracellular antigens secreted into a medium for growing the cells of the Pythium insidiosum in a sterile aqueous solution; and (b) vaccinating the patient with the vaccine.
In further embodiment of the method, the vaccination is subcutaneous. Further still, the patient after vaccination is monitored for a change in a Th1 response and a Th2 response, wherein an increase in the Th1 response and a decrease in the Th2 indicates the patient has developed the Th1 response to the vaccine.
Further still, the present invention provides a method for the treatment of pythiosis in a mammal having pythiosis or prophylaxis against pythiosis in a mammal which comprises (a) providing an injectable vaccine which comprises in a sterile aqueous solution in admixture (i) intracellular cytoplasmic antigens separated from disrupted cells of Pythium insidiosum; and (ii) extracellular antigens secreted into a medium for growing the cells of the Pythium insidiosum; and (b) vaccinating the mammal with the vaccine. Further still, the mammal after vaccination is monitored for a change in a Th1 response and a Th2 response, wherein an increase in the Th1 response and a decrease in the Th2 indicates the patient has developed the Th1 response to the vaccine.
In a further embodiment of the method, the antigens have been provided by (a) growing cells of the Pythium insidiosum in a culture medium and then (i) killing the cells; (ii) separating the killed cells from the culture medium so as to produce a first supernatant comprising the extracellular antigens secreted into the medium; and (ii) disrupting the cells in water to provide the intracellular cytoplasmic antigens in a second supernatant which is separated from the disrupted cells; and (b) separating the extracellular antigens from the first supernatant.
The present invention further provides a method for providing an injectable vaccine for treatment of pythiosis or prophylaxis against pythiosis which comprises (a) growing cells of Pythium insidiosum in a culture medium; (b) separating the cells from a first supernatant of the culture medium which contains extracellular proteins; (c) killing the cells; (d) disrupting the cells in sterile water; (e) separating the disrupted cells from the water to produce a second supernatant containing intracellular proteins; (f) mixing the first supernatant of step (b) with the second supernatant of step (e); (g) separating the combined proteins from the mixture of step (f); (h) mixing the separated proteins in sterile distilled water; and (i) dialyzing the mixture of step (h) to remove low molecular weight components less than 10,000 MW to produce the vaccine.
The present invention further provides a method of testing a response in a mammal to a Pythium insidiosum vaccine including a cell-derived vaccine which comprises monitoring a Th1 response and a Th2 response of the mammal to the vaccine, wherein in mammals which are responding to the vaccine the Th1 response increases and the Th2 response decreases.
In a further embodiment of the method, the vaccine comprises antigens selected from the group consisting of intracellular cytoplasmic antigens separated from disrupted cells of Pythium insidiosum, extracellular antigens secreted into a medium for growing the cells of the Pythium insidiosum, and combination of both.
In a further embodiment of the method, the antigens have been provided by growing cells of the Pythium insidiosum in a culture medium and then killing the cells, separating the killed cells from the culture medium so as to produce a first supernatant comprising the extracellular antigens secreted into the medium, separating the extracellular antigens from the first supernatant, and disrupting the cells in water to provide the intracellular cytoplasmic antigens in a second supernatant which is separated from the disrupted cells.
In a further embodiment of the method, the mammal is in infected with the Pythiosum insidiosum and the vaccine is for immunotherapy or the mammal is not infected with the Pythiosum insidiosum and the vaccine is for prophylaxis.
The present invention further provides a mammal model for testing a Pythium insidiosum vaccine including a cell-derived vaccine which comprises monitoring a Th1 response and a Th2 response of the mammal in the mammal model to the vaccine, wherein in mammals which are responding to the vaccine the Th1 response increases and the Th2 response decreases.
In a further embodiment of the mammal model, the vaccine comprises antigens selected from the group consisting of intracellular cytoplasmic antigens separated from disrupted cells of Pythium insidiosum, extracellular antigens secreted into a medium for growing the cells of the Pythium insidiosum, and combination of both.
In a further embodiment of the mammal model, the antigens have been provided by growing cells of the Pythium insidiosum in a culture medium and then killing the cells, separating the killed cells from the culture medium so as to produce a first supernatant comprising the extracellular antigens secreted into the medium, separating the extracellular antigens from the first supernatant, and disrupting the cells in water to provide the intracellular cytoplasmic antigens in a second supernatant which is separated from the disrupted cells. Preferably, the mammal in the mammal model is a rabbit.
In a further embodiment of the mammal model, the mammal is in infected with the Pythiosum insidiosum and the vaccine is for immunotherapy or the mammal is not infected with the Pythiosum insidiosum and the vaccine is for prophylaxis.
In a further embodiment of any one of the above embodiments of the present invention, the cells have been disrupted by sonication. In further particular embodiments, the Pythium insidiosum is deposited as ATCC 74446; the culture medium is Sabouraud dextrose broth; the cells are killed with thimerosal; the disrupted cells are separated from the culture medium for the cells by centrifugation; the intracellular cytoplasmic antigens in the second supernatant and the extracellular antigens in the first supernatant are mixed to provide a mixture of antigens, precipitating the mixture of antigens with acetone to provide a precipitate, dissolving the precipitate in sterile distilled water to provide a solution of the antigens, and dialyzing the solution of antigens in sterile distilled water to remove low molecular weight components less than 10,000 MW to provide the vaccine; or, any combination thereof.
Further still, in any one of the above embodiments, the present invention provides the vaccines as disclosed in U.S. Pat. Nos. 5,948,413 and 6,287,573, both to Mendoza. Briefly, the injectable vaccine for treatment of pythiosis or prophylaxis against pythiosis which comprises in a sterile aqueous solution an admixture of (a) intracellular cytoplasmic antigens separated from disrupted cells of Pythium insidiosum; and (b) extracellular antigens secreted into a medium for growing the cells of the Pythium insidiosum. 
Preferably, the antigens in the injectable vaccine have been provided by (a) growing cells of the Pythium insidiosum in a culture medium and then (i) killing the cells; (ii) separating the killed cells from the culture medium so as to produce a first supernatant comprising the extracellular antigens secreted into the medium; and (ii) disrupting the cells in water to provide the intracellular cytoplasmic antigens in a second supernatant which is separated from the disrupted cells; and (b) separating the extracellular antigens from the first supernatant.
It is further preferable that the cells have been disrupted by sonication. Further still, it is preferable that the Pythium insidiosum is deposited as ATCC 74446. Further still, it is preferable that the culture medium is Sabouraud dextrose broth. Further still, it is preferable that the cells are killed with thimerosal. Further still, it is preferable that the disrupted cells are separated from the culture medium by centrifugation. Further still, it is preferable that the intracellular cytoplasmic antigens in the second supernatant and the extracellular antigens in the first supernatant are mixed to provide a mixture of antigens, precipitating the mixture of antigens with acetone to provide a precipitate, dissolving the precipitate in sterile distilled water to provide a solution of the antigens, and dialyzing the solution of antigens in sterile distilled water to remove low molecular weight components less than 10,000 MW to provide the vaccine.
It is therefore an object of the present invention to provide a method for treating pythiosis and for prophylaxis against pythiosis in humans and lower animals.
Further, it is an object of the present invention to provide vaccine compositions and methods for the preparation thereof.
Further still, it is an object of the present invention to provide a method for treating pythiosis or prophylaxis against pythiosis which is economical, reliable, and effective.
Further still, it is an object of the present invention to provide a method for monitoring the response of an animal to immunotherapy with the vaccine of the present invention.
Further still, it is an object of the present invention to provide a model system for testing the immune responses to the vaccine of the present invention.
These and other objects of the present invention will become increasingly apparent with reference to the following drawings and preferred embodiments.